This proposal represents a supplemental application to cover additional research not covered by the original application. The overall objective of the original application is to investigate the relationships between the biochemical structures of heparins and their anticoagulant activities. In this proposal we seek to investigate the relationships between anionic density of the heparin and both affinity for individual plasma proteins (antithrombin, thrombin, factor X) and a platelet protein (platelet factor-4) and the effect of heparin on their activities. A combination of fractionation by partition, which separates heparins according to their anionic density, and affinity chromatography on immobilized proteins listed above will be used on different commercial pig-mucosal and beef-lung heparins. Heparin fractions will be characterized with regard to their activities in a series of different anticoagulant and enzymatic assays which measure different functional activities of the heparin molecule. Objectives are to further characterize the structural basis for different activities, to determine structural bases for differences in functional activities, which is responsible for the lack of comparability of different heparins from different tissues and from various commercial sources, and provide for the improved standardization and assay of heparins. In addition, these studies should yield considerable insight into the molecular mechanisms governing the action of heparin and, perhaps, the coagulation system.